The nano-step height reference materials can deliver accurate and traceable nanometer height characteristic parameters. To investigate the fabrication technique of high-quality sub-50 nm step height reference materials in China, a method based on atomic layer deposition combined with wet etching is proposed. The sub-50 nm step height reference materials with a minimum height of only 5 nm are fabricated through process optimization, which achieves the precise control of the step height in the order of the sub-nanometer. The calibration results are traceable to the national meter-defined wavelength reference, and the extended uncertainty is less than 2. 0 nm. Meanwhile, the reference materials has excellent uniformity and stability, and the consistency level of different measurement instruments is quite high. The results show that the nano-step height reference materials can be used for transmission of sub-50 nm height values and comparative measurement between multiple instruments. The prospect of their industrialized production will also provide a perfect metrology guarantee for the semiconductor industry.